The long-range objectives of this application are to precisely control the differentiation of embryonic stem cells for therapeutic screening and treatment of obesity and diabetes. The goal is to achieve an unprecedented understanding of how specific microenvironmental factors commit embryonic stem cells to fully functioning white and brown adipocytes. Obesity is the most rapidly growing disease worldwide and a leading risk factor for diabetes, cardiovascular diseases and some cancer. Adipocytes play a key role in energy imbalance and obesity, wherein white adipocytes store energy in fat and brown adipocytes dissipate energy. Understanding and manipulating white and brown adipogenesis of embryonic stem cells is of paramount importance to the prevention and treatment of obesity. The central hypothesis is that the adipogenic differentiation of embryonic stem cell differentiation is influenced by the combination of microenvironmental factors. A library of stem cell microenvironments built with laser cell printing can be screened and optimized for high efficiency generation of white and brown adipocytes. To accomplish the overall objective, the following Specific AIMs are proposed: 1) to drive embryonic stem cells to become white adipocytes by precise manipulation of the cellular composition, soluble factors, and substrate properties of the stem cell microenvironment;2) to drive embryonic stem cells to become brown adipocytes, by precise manipulation of the stem cell microenvironment;3) to drive existing white adipocytes to transdifferentiate into brown adipocytes through the manipulation of the cellular microenvironment, and vise versa;and 4) to determine if the influences of the microenvironmental factors identified to impact adipogenic embryonic stem cells in 2-D culture are valid when the cellular culture is extended into a 3-D microenvironment. If successful, this project will provide an important platform for understanding of adipogenesis and pharmacological studies of obesity, which will lead to the prevention and treatment of obesity and fat-related diseases, such as diabetes, cardiovascular disease and breast cancer.